Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter.

The promoter of the bean PAL2 gene (encoding phenylalanine ammonia-lyase; EC 4.3.1.5) is a model for studies of tissue-restricted gene expression in plants. Petal epidermis is one of the tissues in which this promoter is activated in tobacco. Previous work suggested that a major factor establishing the pattern of PAL2 expression in tobacco petals is the tissue distribution of a protein closely related to Myb305, which is a Myb-like transcriptional activator from snapdragon. In the present work, we show that Myb305 expression in tobacco leaves causes ectopic activation of the PAL2 promoter. To achieve Myb305 expression in planta, a viral expression vector was used. This approach combines the utility of transient assays with the possibility of direct biochemical detection of the introduced factor and may have wider application for studying the function of plant transcription factors.

Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.

Elicitors of induced disease resistance in postharvest horticultural crops: a brief review

Increasing loss of conventional fungicides due to pathogen resistance and general unacceptability in terms of public and environmental risk have favoured the introduction of integrated pest management (IPM) programmes. Induction of natural disease resistance (NDR) in harvested horticultural crops using physical, biological and/or chemical elicitors has received increasing attention over recent years, it being considered a preferred strategy for disease management. This article reviews the enhancement of constitutive and inducible antifungal compounds and suppression of postharvest diseases through using elicitors. The effect of timing of pre- and/or postharvest elicitor treatment and environment on the degree of elicitation and the potential for inducing local acquired resistance, systemic acquired resistance and/or induced systemic resistance to reduce postharvest disease is discussed. The review highlights that more applied and basic research is required to understand the role that induced NDR can play in achieving practical suppression of postharvest diseases as part of an IPM approach. (C) 2003 Elsevier B.V. All rights reserved.

Stem End blockaged in Cut grevillea 'Crimson Yul-lo' inflorescences

Grevillea 'Crimson Yul-lo' inflorescences have cut flower potential, but their vase life is short. End of vase life is characterized by early wilting. The possibility of physiologically mediated stem end blockage was investigated. Hydraulic conductance of 2 cm long stem end segments declined rapidly and remained lower throughout vase life than that of 2 cm long stem segments from immediately above. Recutting daily to remove basal 2 cm stem ends increased solution uptake, delayed declines in inflorescence water potential and water content, and improved inflorescence vase life. S-carvone is a potential inhibitor of wound related suberin formation, via inhibition of phenylalanine ammonia-lyase. Vase solution treatments with S-carvone (0.318 and 0.636 mM) delayed the decline in hydraulic conductance of basal 2 cm long stem end segments and decreases in vase solution uptake and relative fresh weight of cut stems, and extended vase life. Treatments with the catechol oxidase inhibitor 4-hexylresorcinol (2.5-10 mM) also delayed stem end blockage. These findings suggest that stem end blockage in cut G. 'Crimson Yul-lo' stems is physiologically mediated. (C) 2006 Elsevier B.V. All rights reserved.

Potencial do óleo de Aloysia citriodora Palau no controle de fitopatógenos e na indução de resistência ao tombamento de plântulas de feijão, pepino e beterraba

The crops are affected by pests and diseases that decrease productivity. Among them are the damping off of seedlings that can occur in pre and post-emergence. In bean crops, cucumber and beet these diseases occur, being caused by various pathogens, especialy fitopathogenic fungi. Several measures are used for the controle of such diseases, among them, is the chemical seed treatment fungicides. However, society has become increasingly concerned about the quality and food and environmental contamination, generation a growting search for sensitive products to humans and the environment. The use of essential oils to control plant pathogens is an example of alternative tested by science in the search for less aggressive technologies. This study aimed to evaluate the efficiency of the use of essential oil Aloysia citriodora, in control of pathogens causing damping off in beans, cucumber and beet. This thesis was divided in four chapters, the introductory first, and the other addressing the control of Pythium sp. in beans, Sclerotinia sclerotiorum on cucumber, and Fusarium sp. on beet. The methodology consisted of four experiments in each pathosystem, with all the work done at the Federal Technological University of Parana, Campus Dois Vizinhos. In the first experiment evaluated the fungistatic and fungicidal effect of the essential oil of A. citriodora on PDA in vitro in mycelial growth of pathogens studied. In the second experiment evaluated the in vitro effect of essential oil concentrations of A. citriodora in BD medium on microscope slides, on the germination of sporangia Pythium sp. and conidia Fusarium sp., and in Petri dishes with PDA medium, the sclerotia germination speed index of S. sclerotiorum. In the third experiment, we evaluated in germination test in paper roll (PR), the phytotoxic effect or not the use of essential oil concentrations of A. citriodora in dry bean seed, cucumber and beet. The variables used to assess this experiment were the germination percentage, mediun green mass per plant and average length of seedlings. In the fourth experiment we assessed the effect of treating bean seeds, cucumber and beet with essential oil contents of A. citriodora, seeds in their subsequent substrates contamined with pathogens studied, Pythium sp., S. sclerotiorum and Fusarium sp. In this experiment we used the following variables: percentage of emergence, percentage of post-emergence damping off, green average mass per plant, average length per plant and biochemical analyzes. The biochemistry of plant tissues evaluated were as follows: protein content, enzymatic activities of peroxidases, phenylalanine ammonia-liase (PAL), chitinases and β-1,3-glucanases. The in vitro results show that the essential oil has fungistatic and fungicidal effect on mycelial growth, on sporangia germination, conidia and sclerotia of the pathogens studied in this work, wich may be related to its major components, citral and limonene. The oil also exhibits low phytotoxicity to seeds of the species studied, only in beans decreases germination in most studied dosage (0,25%), cucumber also in the higher dosage (0,25%) reduce the length of seedlings, and beet there were no negative effects to the seedlings. In the test in substrate contaminated with the pathogens, the use of essential oil: increased germination and decreased post emergence damping off of beans seedlings; at a concentration of 0,0625% decreases post emergence damping off in cucumber. In biochemical analyzes found an increase in the enzymatic activity of peroxidases and β-1,3-glucanases on beans, and glucanases on cucumber, and increased enzyme activity of peroxidases on beet, showing action in resistance induction at damping off.

Desempenho de fosfitos de potássio no manejo de mofo branco em soja

Soybean plays an important role in the Brazilian agriculture being one of the products most exported by the country. Its yield may be affected by diseases such as white mold, caused by the fungus Sclerotinia sclerotiorum Lib. de Bary, which, under favorable field conditions prevents the crop of expressing all its productive potential. The fungus is cosmopolitan and infects more than 400 species of plants. This disease is difficult to control, and the use of chemicals has not been sufficient to avoid significant losses, thus, this products are expensive and may cause environmental damage. Alternative methods, such as foliar fertilizers based on potassium phosphite, can also be used in the management of this disease. In this context, this work aimed to study different sources of potassium phosphite and its effects in the control of white mold in soybeans, as well as the time of application in culture, its action in inducing plants defense responses and/or its influence over the seeds quality. The effect of phosphites, over the pathogen, was evaluated in vitro, on mycelial inhibition, the mass of dry mycelium and germination of sclerotia. In all tests, the following phosphites were utilized: Phosphite A (P2O5-40%; K2O-20% - 1 L/ha); Phosphite B (P2O5-40%; K2O-28% - 1 L/ha); Phosphite C (P2O5-40%; K2O-20% - 1 L/ha) e Phosphite D (P2O5-30%; K2O-20% - 2,4 L/ha). At the induction of resistance tests were evaluated the synthesis of phytoalexin in soybean cotyledons and the enzymes FAL and POX evaluated in seedlings in growing chamber, sprayed with phosphites and the fungicide fluazinam. Field experiment was carried out at Coronel Domingos Soares-PR, in the 2012/2013 season, in an area with natural infestation of the pathogen. Soybean cultivar BMX Active was no-till seeded with 0,5m between rows. The experimental was laid out as a factorial 5 x 4 scheme (treatment x application time). Phosphites sources were used, as described above, and water was sprayed in the control treatment. Treatments were applied at four different growth stages: V4, V4 + R1, R1 and R2 at the rates recommended by the manufacturer. Soybean yield components and seeds and health and physiological quality were evaluated after harvesting. None of the tested phosphites affected mycelial growth and sclerotia germination or influenced phytoalexin synthesis. Phosphites C and D stood out due to an increasing in the phenylalanine ammonia-lyase activity 48 hours after its inoculation. These same products also induced the synthesis and peroxidases and phosphite C kept the levels of this enzyme elevated up to 72 hours after inoculation. At the field trials, phosphites C and D stood out in the control of white mold. There was no significant interaction of potassium phosphite on physiological and sanitary quality of the seeds.

Postharvest light treatments increase skin blush in mango fruit

Skin colour is an important quality parameter that influences mango fruit marketability. The mango industry is interested in controlled induction of skin blush in mangoes. It is desirable to understand the control of anthocyanin accumulation in mango skin. Among environmental factors known to induce anthocyanin accumulation in plants, light is the most studied. Light exposure induces pigmentation in various fruits, including apple, strawberry and grape. The effect of different light qualities on skin blush in mango fruit has received relatively little attention. The objective of this study was to assess anthocyanin accumulation and blush in response to blue, red and far red light from light-emitting diodes (LEDs) as applied to harvested mango fruit skin during storage at 12°C. Except for red light, the other wavelengths induced anthocyanin accumulation and skin blush as compared to the dark control treatment. Anthocyanin concentration and a∗ values were highest in blue light exposed fruit skin. This wavelength enhanced phenylalanine ammonia lyase activity in the mango skin, which may be associated with increased pigmentation. LED light treatment did not affect other fruit quality parameters at 21 days of storage, including firmness, total soluble solids and titratable acidity. Overall, the findings suggest that postharvest treatment with blue light can induce skin blush in mango fruit, which potentially may enhance their commercial value.

The Decisive Step in Betaxanthin Biosynthesis Is a Spontaneous Reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.

Biosynthetic production of curcuminoids

Curcuminoids are natural phenylpropanoids from plants that have been reported as potential cancer-fighting drugs. Nevertheless, these compounds present a poor bioavailability. Cellular uptake is low and curcuminoids are quickly metabolized once inside the cell, requiring repetitive oral doses to achieve an effective concentration for therapeutic activity [1]. Herein, we report an engineered artificial pathway for the production of curcuminoids in Escherichia coli. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used and 188 µM (70 mg/L) of curcumin was obtained from ferulic acid [2]. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used [3]. Caffeoyl-CoA 3-O-methyl-transferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 µM of curcumin were produced, respectively. This is the first report on the use of DCS and CURS1 in vivo to produce curcuminoids. In addition, curcumin, the most studied curcuminoid for therapeutic purposes and considered in many studies as the most potent and active, was produced by feeding tyrosine using a pathway involving caffeic acid. We anticipate that by using a tyrosine overproducing strain, curcumin can be produced in E. coli without the need of adding expensive precursors to the medium, thus decreasing the production cost. Therefore, this alternative pathway represents a step forward in the heterologous production of curcumin using E. coli. Aiming at greater production titers and yields, the construction of this pathway in another model organism such as Saccharomyces cerevisiae is being considered.

Synthetic biology approaches to engineer new pathways for the production of plant secondary metabolites

Secondary metabolites from plants are important sources of high-value chemicals, many of them being pharmacologically active. These metabolites are commonly isolated through inefficient extractions from natural biological sources and are often difficult to synthesize chemically. Therefore, their production using engineered organisms has lately attracted an increased attention. Curcuminoids, an example of such metabolites, are produced in Curcuma longa and exhibit anti-cancer and anti-inflammatory activities. Herein we report the construction of an artificial biosynthetic pathway for the curcuminoids production in Escherichia coli. Different 4-coumaroyl-CoA ligases (4CL) and polyketide synthases (diketide-CoA synthase (DCS), curcumin synthase (CURS) and curcuminoid synthase) were tested. The highest curcumin production (70 mg/L) was obtained by feeding ferulic acid and with the Arabidopsis thaliana 4CL1 and C. longa DCS and CURS enzymes. Other curcuminoids (bisdemethoxy- and demethoxycurcumin) were also produced by feeding coumaric acid or a mixture of coumaric and ferulic acids, respectively. Curcuminoids, including curcumin, were also produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase and 4-coumarate 3-hydroxylase were used. Caffeoyl-CoA O-methyltransferase was used to convert caffeoyl-CoA to feruloyl-CoA. This pathway represents an improvement of the curcuminoids heterologous production. The construction of this pathway in another model organism is being considered, as well as the introduction of alternative enzymes.

The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

Quitosana na indução de resistência ao tombamento de plântulas de espécies olerícolas e no controle de fitopatógenos in vitro

Induction of resistance is defined as the activation of a state of resistance against diseases which is induced systemically in plants by the use of biotic or abiotic agents without any modification of the plant genome, occurring non-specific way, by activating genes coding for various plant defense responses. Chitosan is a polymer derived from the deacetylation of chitin, which is found in large quantities in crustacean shell, and studied with the potential to control plant pathogens, both by its direct fungistatic action, as the ability to induce protection of plants, indicating the presence of molecules of elicitoras characteristics. Three experiments with objective of evaluating the potential of chitosan in the seedling resistance induction were developed, beet (Beta vulgaris) seeds, cucumber (Cucumis sativus) seeds and tomato (Solanum lycopersicum) seeds, and the control of Fusarium sp., Rhizoctonia solani K¨uhn e Pythium sp. in vitro conditions. The experimental design was completely randomized, with four replications. Beet seeds, tomato and cucumber were submerged in chitosan solution for 20 minutes, in concentrations of 0.25, 0.5, 1 and 2% in the control and distilled water. Seeds were sown in trays containing Plantmax Florestalr substrate sterilized and inoculated with Fusarium sp., Rhizoctonia solani K¨unh and Pythium sp., respectively for the three cultures. The experiment was conducted for 14 days in growth chamber with controlled temperature (25 C 2 C), light (12 hour photoperiod) and humidity (70% 10%). The evaluations were seed emergency, seedling damping-off, seedling length, fresh weight and activity of the enzymes phenylalanine amˆonia-liase (PAL), chitinase and b-1,3-glucanase. It was also rated the mycelial growth of Fusarium sp., Pythium sp. and R. solani on P.D.A. (Potato-Dextrose and Agar) culture medium containing chitosan at the same concentrations evaluated in seeds. For beet growing, seed treatment with chitosan presented higher emergence and the length of the seedlings, and reduced the percentage of tipping. Treatment with chitosan activated the systemic acquired resistance with expression of chitinase and b-1,3-glucanase enzymes. For the tomato crop in chitosan concentration of 0.25% favored the emergency of seedlings, reduced the incidence of tipping and activated the PAL enzymes, chitinase and b-1,3-glucanase. In cucumber on the concentration of up 0.5% favored seedlings emergence and reduces the incidence of tipping. Chitosan activated the PAL enzymes and b-1,3-glucanase. Chitosan also presented fungistatic action on the initial growth of Pythium sp. and R. solani in vitro conditions, however, such action did not prevail until the end of the experiment. To Fusarium sp. the concentration of chitosan resulted in the reduction of mycelial growth in vitro.

Hyperammonemia: regulation of argininosuccinate synthetase and argininosuccinate lyase genes in aggregating cell cultures of fetal rat brain.

Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis. Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). The regulation of AS and AL genes during hyperammonemia is unknown in the brain. We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia. Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl. Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

Six new cases confirm the clinical molecular profile of complete combined 17α-hydroxylase/ 17,20-lyase deficiency in Brazil

In 2004, Costa-Santos and cols. reported 24 patients from 19 Brazilian families with 17α-hydroxylase deficiency and showed that p.W406R and p.R362C corresponded to 50% and 32% of CYP17A1 mutant alleles, respectively. The present report describes clinical and molecular data of six patients from three inbred Brazilian families with 17α-hydroxlyse deficiency. All patients had hypogonadism, amenorrhea and hypertension at diagnosis. Two sisters were found to be 46,XY with both gonads palpable in the inguinal region. All patients presented hypergonadotrophic hypogonadism, with high levels of ACTH (> 104 ng/mL), suppressed plasmatic renin activity, low levels of potassium (< 2.8 mEq/L) and elevated progesterone levels (> 4.4 ng/mL). Three of them, including two sisters, were homozygous for p.W406R mutation and the other three (two sisters and one cousin) were homozygous for p.R362C. The finding of p.W406R and p.R362C in the CYP17A1 gene here reported in additional families, confirms them as the most frequent mutations causing complete combined 17α-hydroxylase/17,20-lyase deficiency in Brazilian patients.

Electronic properties of liquid ammonia: A sequential molecular dynamics/quantum mechanics approach

The electronic properties of liquid ammonia are investigated by a sequential molecular dynamics/quantum mechanics approach. Quantum mechanics calculations for the liquid phase are based on a reparametrized hybrid exchange-correlation functional that reproduces the electronic properties of ammonia clusters [(NH(3))(n); n=1-5]. For these small clusters, electron binding energies based on Green's function or electron propagator theory, coupled cluster with single, double, and perturbative triple excitations, and density functional theory (DFT) are compared. Reparametrized DFT results for the dipole moment, electron binding energies, and electronic density of states of liquid ammonia are reported. The calculated average dipole moment of liquid ammonia (2.05 +/- 0.09 D) corresponds to an increase of 27% compared to the gas phase value and it is 0.23 D above a prediction based on a polarizable model of liquid ammonia [Deng , J. Chem. Phys. 100, 7590 (1994)]. Our estimate for the ionization potential of liquid ammonia is 9.74 +/- 0.73 eV, which is approximately 1.0 eV below the gas phase value for the isolated molecule. The theoretical vertical electron affinity of liquid ammonia is predicted as 0.16 +/- 0.22 eV, in good agreement with the experimental result for the location of the bottom of the conduction band (-V(0)=0.2 eV). Vertical ionization potentials and electron affinities correlate with the total dipole moment of ammonia aggregates. (c) 2008 American Institute of Physics.